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First Report of Alternaria alternata Causing Leaf Spot on Rumex crispus in Uruguay

Kaspary, T. E., García, M. A., Stewart, S., Casaroto, G., Ramos, R. F., Bellé, C.
Plant disease 2019 v.103 no.8 pp. 2139
Alternaria alternata, DNA, DNA-directed RNA polymerase, Rumex crispus, alternative hosts, beak, conidia, conidiophores, crops, ethanol, fungi, genes, glyceraldehyde-3-phosphate dehydrogenase, grasslands, hyphae, leaf spot, leaves, mycelium, nutrients, nutritive value, pathogenicity, peptide elongation factors, weeds, Uruguay
Rumex crispus L. (curled dock), a member of the family Polygonaceae, is considered an important weed in grasslands and crops (Hejcman et al. 2012). Curled dock decreases yield and nutritive value of different species by competition for space, water, and nutrients (Zaller 2004). In addition, it may be an alternative host of pests and diseases of crops (Abbasi et al. 2018). In October 2018, R. crispus plants with severe spotting on their leaves were observed in a field at La Estanzuela, in Colonia, a department in southwestern Uruguay. Symptoms were small, circular, light brown spots that eventually turned into irregular, dark brown lesions, although a few remained circular with concentric rings. Leaf lesion samples were surface sterilized (70% ethanol for 30 s, 1% NaClO for 2 min, rinsed three times in sterile water, dried on sterilized filter paper) and placed on potato dextrose agar (PDA). To identify the causal fungus, morphological analysis was conducted, followed by molecular characterization through the amplification and sequencing of the internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the second largest subunit of RNA polymerase II (RPB2), and the translation elongation factor (TEF-1α) gene regions, using the method described by Woudenberg et al. (2013). Colonies were round, composed of cottony mycelium of dark olivaceous hyphae. Conidiophores were septate, light-to-olive golden brown, with a conidial scar where conidia were produced in long chains. Conidia (n = 50) were obclavate to obpyriform, golden brown, with a cylindrical or coniform beak at the tip, and measured 25.9 to 61.5 μm long × 12.5 to 16.5 μm wide with two to six transverse and one to three longitudinal septa. Sequences of the studied DNA regions were submitted to GenBank, and accession numbers were received (ITS, MK635345; GAPDH, MK639185; RPB2, MK645322; TEF-1α, MK639186). BLAST searches showed 99 to 100% identity with the existing sequences (including ex-type CBS 916.96) of Alternaria alternata (Fr.) Keissl. (1912) (ITS, AF347031; GAPDH, AY278808; RPB2, KC584375; TEF-1α, KC584634). To confirm Koch’s postulates, six plants of R. crispus were inoculated with a suspension of 10⁶ conidia/ml, whereas six plants to which distilled water was applied were considered as controls. All plants were enclosed in plastic bags and incubated in a growth chamber at 25 ± 2°C for 24 h with a 12-h photoperiod. At the end of the 24-h period, plastic bags were removed. Ten days after inoculation, leaves displayed symptoms similar to those observed in the field, whereas controls remained symptomless. The fungus reisolated from inoculated leaves had the same morphological and molecular traits as the initial isolate. Pathogenicity tests were carried out two times. Our findings highlight the relevance of R. crispus as a reservoir for an important crop pathogen as well as a major crop competitor. To our knowledge, this is the first report on A. alternata affecting R. crispus plants in Uruguay.